Post-menopausal women are especially vulnerable to ischemic insult, and ~80,000 more cardiovascular deaths occur yearly in aged women than in aged men. Conflicting data exist on the role of estrogen (E2) in modulating ischemic tolerance (IT), due in part to the failed efficacy of E2 replacement to improve IT. Protective signaling pathways in the heart known to be influenced by E2 action include (PI3K)-Akt, eNOS and GSK3-2. Collectively, these signals impact mitochondrial events critical to cell survival to limit necrotic and apoptotic cell death. Here, we propose to test the novel paradigm that maladaptive estrogen receptor (ER) expression in the aged heart leads to a vicious cycle of increased oxidative stress (ROS) and ischemia/reperfusion (I/R injury), while selective engagement of non-genomic ER pathways can rescue this phenotype. We further propose that non-genomic ER signaling results in protein kinase C5 (PKC5) activation, a well-known mediator of cardioprotection. The following specific aims will be tested: 1) determine which ER subtype(s) reduce IT in aged, gonadectomized female rats, 2) determine the molecular mechanism(s) by which ERs alter ROS (ONOO- and O-2) through eNOS uncoupling, post-translational modification (PTM) or the PI3- Akt-GSK-32 axis, 3) determine the role of PKC5 in rapid ER signaling, and 4) identify novel mitochondrial PKC5 binding targets following non-genomic ER activation. Using a model of global ischemia, we will employ specific ER and PKC5 agonists and EPR spectroscopy to assess ROS. Apoptosis will be assessed through in vivo coronary ligation, western blotting for cellular ER subtype targeting and downstream signals, qRT- PCR for ER- mediated gene expression, and multiple reaction monitoring to assess PTMs of ERs. A targeted, state-of-the- art proteomic iTRAQ 8plex approach and LC MS/MS will be used to identify novel cardiac mitochondrial PKC5 protein partners and phosphorylation. We propose that non-genomic activation of ERs can compensate for E2 deficiency in aged female hearts, resulting in improved IT. The confirmation of our hypothesis will strongly support new targets for the treatment of acute coronary syndrome in the aging population and address the potential use of ER/PKC5 therapeutics as viable approaches to improve IT in post-menopausal women. PUBLIC HEALTH RELEVANCE: The leading cause of death among persons aged 75 years or older is heart disease, and deaths due to heart disease have actually increased by 60% from 1970 to 2002 among persons aged 80 years or older. Age is also the leading risk factor for the development of heart failure (CHF) and more Americans aged 65 and older are treated and discharged from hospitals with CHF than with any other diagnosis. Interestingly, mortality rates due to CHF are significantly greater in aged women vs aged men. CVD rates and associated mortality are also significantly greater in aged women when compared to aged men, and data from large scale clinical trials suggest that estrogen replacement therapy is ineffective in reducing, and may actually increase CVD outcomes. Collectively, this expanding aged demographic will undoubtedly require increased allocation of medical resources, raising the economic burden of health care costs for all Americans. The cellular mechanisms by which estrogen exerts effects in the aged heart, however, are poorly understood. The experiments associated with this proposal are expected to identify novel protein targets previously unstudied with estrogen deficiency. If our hypothesis on estrogen receptors is correct, new therapeutic targets for the treatment of ischemic cardiovascular disease in the aged population will be realized, i.e. use of estrogen receptor therapeutics as viable approaches to improve ischemic tolerance in aged women.